Gene therapy aims to correct defective genes that underlie the development of diseases. A common approach to addressing this issue involves the delivery of a normal gene to the nucleus. This gene may then be inserted into the genome of the targeted cell or may remain episomal. Delivery of a corrective gene to a subject's target cells can be carried out via numerous methods, including the use of viral vectors. Among the many viral vectors available (e.g., retrovirus, lentivirus, adenovirus, and the like), adeno-associated virus (AAV) is gaining popularity as a versatile vector in gene therapy.
Adeno-associated virus (AAV) is a member of the parvoviridae family. The AAV genome is composed of a linear single-stranded DNA molecule which contains approximately 4.7 kilobases (kb) and consists of two major open reading frames encoding the non-structural Rep (replication) and structural Cap (capsid) proteins. Flanking the AAV coding regions are two cis-acting nucleotide inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can fold into hairpin structures that function as primers during initiation of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown to be necessary for viral integration, rescue from the host genome, and encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).
Vectors derived from AAV are particularly attractive for delivering genetic material because (i) they are able to infect (transduce) a wide variety of non-dividing and dividing cell types including muscle fibers and neurons; (ii) they are devoid of the virus structural genes, thereby eliminating the natural host cell responses to virus infection, e.g., interferon-mediated responses; (iii) wild-type viruses have never been associated with any pathology in humans; (iv) in contrast to wild type AAVs, which are capable of integrating into the host cell genome, replication-deficient AAV vectors generally persist as episomes, thus limiting the risk of insertional mutagenesis or activation of oncogenes; and (v) in contrast to other vector systems, AAV vectors do not trigger a significant immune response (see ii), thus granting long-term expression of the therapeutic transgenes (provided their gene products are not rejected). AAV vectors can also be produced at high titer and intra-arterial, intra-venous, or intra-peritoneal injections allow gene transfer to significant muscle regions through a single injection, at least in rodents (Goyenvalle et al., 2004; Fougerousse et al., 2007; Koppanati et al., 2010; Wang et al., 2009). AAV vectors also have multiple advantages over plasmid DNA with respect to delivering genetic material. For instance, expression of heterologous genes from plasmids is short-term, plasmids are usually of greater size, and plasmids need to be physically manipulated in order to be delivered into cells (e.g., microinjection, transfection, electroporation). Furthermore, plasmid transfer of genes such as dystrophin elicits an immune response in the host and is associated with low efficiency (Romero et al., 2004).
However, the use of conventional AAV as a gene delivery vector is also associated with numerous drawbacks. First, a significant proportion of potential recipients of such therapy are already seropositive to a given type of AAV vector (Boutin et al., 2010). Second, in some individuals AAV vectors may elicit a mild host immune response likely mediated by the viral capsid or processing impurities. In this context, use of immunosuppressants to control the immune response is only a temporary measure, as once a subject is taken off these immunosuppressants, the immune response returns (Lorain et al., 2008). Perhaps the main drawback associated with AAV is its limited viral packaging capacity of about 4.5 kb of heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010).
Different approaches using dual-vector strategies have been attempted to expand AAV vector packaging capacity, such as trans-splicing (ts) and overlapping (ov) AAV vector systems designed to deliver large therapeutic genes to target cells. For instance, Lostal et al. bypassed the size limitation with respect to dysferlin cDNA, which cannot be directly incorporated into an AAV vector for gene transfer into muscle, by splitting the dysferlin cDNA at the exon 28/29 junction and cloning it into two independent AAV vectors carrying appropriate splice sequences (Lostal et al., 2010). Yet, even these approaches suffer from inherent inefficiency. Thus, the small packaging capacity remains a major limitation in AAV gene therapy. Removal of the capsid has not been considered a way by which the packaging capacity could be overcome because the capsid is considered essential to permit vector entry into the cell.
Another notable drawback is that AAV-mediated gene expression is relatively slow, given that single-stranded AAV DNA must be converted to double-stranded DNA prior to heterologous gene expression. While attempts have been made to circumvent this issue by constructing double-stranded DNA vectors, this strategy further limits the size of the transgene expression cassette that can be integrated into the AAV vector (McCarty, 2008; Varenika et al., 2009; Foust et al., 2009). Furthermore, effective AAV-mediated gene therapy in a growing organ can lose its effect due to episomal DNA dilution in dividing cells (Cunningham et al., 2009).
The present invention addresses some or all of the aforementioned drawbacks associated with AAV vectors by providing recombinant AAV0 (“rAAV0”) vectors for in vitro, ex vivo, or in vivo delivery of exogenous DNA sequences to a cell, tissue, organ, or subject.